Plant breeding is the science that utilizes crosses between individuals with different genetic constitutions. The resulting recombination of genes between different lines, species or genera produces new hybrids from which desirable traits are selected. Methods employed to develop new varieties or species depend on whether a crop plant reproduces sexually or asexually. Since maize is a sexually reproducing plant, techniques for controlled pollination are frequently employed to obtain new hybrids.
A significant technological breakthrough in maize breeding was the discovery that crossing inbred lines resulted in a hybrid with greatly enhanced vigor. Inbred lines are obtained from self-pollination and selection of homozygous plants for several generations until a pure line descended by self-pollination from an apparently true-breeding plant is obtained. The purpose of inbreeding is to fix desirable characters in a homozygous condition in order that the line may be maintained without genetic change. Inbred lines with desired traits are then crossed to produce commercial hybrids. Yields from hybrid maize seed are much greater than average yields of inbreds and open-pollinated varieties.
Maize is a monoecious grass, i.e. it has separate male and female flowers. The staminate, i.e. pollen-producing, flowers are produced in the tassel and the pistillate or female flowers are produced on the shoot. Pollination is accomplished by the transfer of pollen from the tassel to the silks. Since maize is naturally cross-pollinated, controlled pollination, in which pollen collected from the tassel of one plant is transferred by hand to the silks of another plant, is a technique used in maize breeding. The steps involved in making controlled crosses and self-pollinations in maize are as follows: (1) the ear emerging from the leaf shoot is covered with an ear shoot bag one or two days before the silks emerge to prevent pollination; (2) on the day before making a pollination, the ear shoot bag is removed momentarily to cut back the silks, then is immediately placed back over the ear; (3) on the day before making a pollination, the tassel is covered with a tassel bag to collect pollen; (3) on the day of pollination, the tassel bag with the desired pollen is carried to the plant for crossing, the ear shoot bag is removed and the pollen dusted on the silk brush, the tassel bag is then immediately fastened in place over the shoot to protect the developing ear. Wild relatives of crop plants are an important source of genetic diversity and genes well adapted to many different stresses. The wild relatives of maize include annual teosinte (Zea mexicana), perennial teosinte and Tripsacum. Zea diploperennis (hereafter referred to as diploperennis), is a diploid perennial teosinte. A previously unknown wild relative of maize, it was discovered on the verge of extinction in the mountains of Jalisco, Mexico in 1979. Diploperennis, like annual teosinte, is in the same genus as maize, has the same chromosome number (n=10), and hybridizes naturally with it. Tripsacum is a more distant relative of maize with a different haploid chromosome number (n=18). The progeny of (maize X Tripsacum) obtained by artificial methods are all male sterile and have limited female fertility when pollinated by maize pollen. Cytogenetic studies of maize-Tripsacum hybrids show partial chromosome pairing and homology between segments of Tripsacum and maize chromosomes (Maguire 1961, 1963; Chaganti 1965; Gallnat 1974). In spite of strong cross-incompatibility, the fact that maize and Tripsacum chromosomes can occasionally pair enables limited transfer of Tripsacum genes into maize. Attempts to make the corollary cross, i.e. between Tripsacum and teosinte, however, have heretofore failed to produce viable plants (Tantravahi 1968; deWet and Harlan 1978).
Plant breeders acknowledge Tripsacum has significant potential for improving corn by expanding its genetic diversity (Gallnat 1977; Cohen and Galinat 1984; Poehlman 1986). The limited fertility of maize-Tripsacum hybrids presents a significant biological barrier to gene flow between these species. Successful introgression of Tripsacum genetic material into maize heretofore has required years of complicated, high risk breeding programs that involve many backcross generations to stabilize desirable Tripsacum genes in maize. According to Kindiger and Beckett: "Tripsacum may be expected to contain valuable agronomic characters that could be exploited for the overall improvement of maize . . . An effective procedure to transfer Tripsacum germ plasm into maize has been needed by maize breeders and geneticists for many years" (1990, p. 495). Beneficial traits that may be derived from Tripsacum include heat and drought tolerance (Reeves and Bockholt 1964), elements of apomixis, increased heterosis (Reeves and Bockholt 1964; Cohen and Galinat 1984), resistance to corn root worm (Branson 1971), corn leaf aphid (Branson 1972), northern and southern leaf blight, common rust, anthracnose, fusarium stalk rot and Stewart's bacterial blight (Bergquist 1977, 1981; deWet 1979).
(Zea mays X Tripsacum) plants have unreduced gametes with 28 chromosomes, one set of 10 Zea chromosomes and one set of 18 Tripsacum chromosomes. There has been one report of a successful reciprocal cross of Tripsacum pollinated by maize in which embryo culture techniques were used to bring the embryo to maturity. The plants were sterile (Farquharson 1957). This (Tripsacura X maize) plant was employed by Branson and Guss (1972) in tests for rootworm resistance in maize-Tripsacum hybrids. When the (maize X Tripsacura) hybrid has been crossed with either annual teosinte or diploperennis, a trigenomic hybrid has been produced that has a total of 38 chromosomes; 10 from maize, 18 from Tripsacum and 10 from teosinte. The resulting trigenomic plants were all male sterile and had a high degree of female infertility (Mangelsdorf 1974; Galinat 1986).
Transformation, a technique from molecular biology, now offers opportunity for the asexual transfer of genes that heretofore could only be achieved by crossing different plant strains. In order for breeders to employ gene transfer via transformation, they first have to be able to achieve plant regeneration from calli or protoplasts. Although transformation has been successfully performed in maize (Gordan-Kamm et al. 1990), there is limitation in developing transgenic maize due to the difficulties of plant regeneration from maize protoplasts (Potrykus 1990). The problem is there are very few maize lines that can be successfully regenerated from maize protoplasts. In order for transformation to be useful for commercial hybrid seed production, it will be necessary to have inbred lines amenable to the transgenic process that can be regenerated by tissue culture.
Rootworms, Diabrotica spp., are a serious agricultural pest. Reduction in corn yields due to corn rootworm damage ranges from 13 to 16 bushels per acre which is approximately 10 to 13%. Costs of insecticide treatments and crop losses are estimated at $1 billion per year (Metcalfe 1986). Rootworm larvae feed on the root system of corn for several weeks passing through three instars. This is the most destructive stage and causes reduced yields through damage to the root system or indirectly from lodging which makes plants difficult to harvest. Adult beetles feed on the aerial parts of the corn plant including the pollen, silks and leaves (Branson et al. 1975).
Zea diploperennis is an acceptable larval host for several Diabrotica species. Feeding scars and leaf damage have been recorded for plants growing in the wild in Jalisco, Mexico, and laboratory screening revealed diploperennis has no antibiotic effect on rootworm larvae (Branson and Reyes 1983). Tripsacum dactyloides, however, has been shown to exhibit a high degree of resistance to corn rootworm (Branson 1971). Screening of intergeneric hybrids between T. dactyloides and Zea mays showed (maize X Tripsacuraum) was susceptible; whereas, (Tripsacura X maize) exhibited resistance (Branson and Guss 1972). The authors proposed two explanations: (1) resistance is inherited through the cytoplasm, or (2) the genes for resistance occur on lost Tripsacum chromosomes in (maize X Tripsacum) plants.
Polyploidy refers to all natural and induced variations in chromosome number. Many cultivated crop species have evolved in nature as polyploids. One way polyploid plants arise is by combining chromosome sets from two or more species which is referred to as allopolyploidy. An allopolyploid, i.e. a plant in which the total chromosome complement of two other species is combined to form a fertile species hybrid, is referred to as an amphiploid. A plant breeding method to transfer genes across a barrier of reproduction isolation is via bridging species derived from an amphiploid. This type of introgressive hybridization produces convergence between previously more distinct species. It may result in the appearance of types that are new species intermediate to their more divergent and distinct parents. Bridging species derived from crosses between two parents with different chromosome numbers are frequently characterized by a new chromosome number. The change in chromosome complement and/or rearrangements in chromosome structure may overcome the inability of chromosomes to pair that causes infertility and often prevents the success of wide crosses.
Two wild grasses, Zea diploperennis and Tripsacum dactyloides have been crossed to produce a novel hybrid referred to as Tripsacorn, proposed botanical name Zea indiana. A bridging mechanism to transfer Tripsacum genes into maize is provided by Tripsacorn which is cross-fertile with maize. It promises to improve corn by imparting numerous beneficial characteristics including pest resistance and drought tolerance.
Based on proposed taxonomic relationships between Zea and Tripsacura and the results of prior crosses between them, the success of the crosses between Zea diploperennis and Tripsacura resulting in fully fertile plants with chromosome numbers of 2n=20 and 2n=18 could not have been predicted. The reduction in chromosome number in the interspecific cross is unexpected based on prior art. The fertility of plants resulting from the cross made both ways is also unexpected. Tripsacum and diploperennis have chromosomes that are very similar architecturally in length and their diminutive, terminal knobs that appear at one or both ends of many of the chromosomes in both species. The small terminal knobs in these species are distinct from the large internal knobs that characterize the chromosomes of corn and annual teosinte. As evidenced by cross fertility and chromosome number, the similarities in the chromosome structure of Tripsacum and diploperennis evidently promote a greater degree of pairing and enable the unexpected success of this cross.
The unexpected fertility of this hybrid, and its cross-fertility with maize, is of great value because it conveys opportunity for directly crossing with maize. Tripsacorn provides a mechanism for importing Tripsacum genes into maize in one generation by natural breeding techniques. Since Tripsacum is the female parent in this cross, it provides unique opportunity for transferring Tripsacum cytoplasmic genes into maize.
Insect resistance derived from crossing Tripsacorn with maize has been demonstrated experimentally. In a series of bioassays, seedlings from (maize X Tripsacorn) infested with western corn rootworm, Diabrotica virgifera Le Conte, showed clear evidence for rootworm resistance. This was corroborated by comparison with maize controls and (maize X Sun Dance) plants, both of which were susceptible, as indicated by considerable root damage or death.